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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Glp guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Diethylentriamine R
- Physical state: liquid
- Analytical purity: The identity of the test substance was confirmed...........

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared form sprague Dawlay rat livers after Aroclor 1254 activation
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500, 5000 µg/plate (SPT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see details
Details on test system and experimental conditions:

Standard Plate Test
Salmonella typhimurium

Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:

0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
Or
0.5 mL phosphate buffer (without metabolic activation)

After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. ( 5), with the exception of the amino acid solution, which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (saline solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.
In each experiment 3 test plates per dose or per control were used.

Positive controls:
2-Aminoanthracene: 2.5 µg/plate for strains: TA 1535, TA 100, TA 1537, TA 98 ( with S9 mix)
60 µg/plate for strain: E. Coli WP2 uvrA ( with S9 mix)
N-methyl-N`-nito-N-nitrosoguanidine : 5 µg/plate for strains: TA 1535, TA 100 (without S9 mix)
4-nitro-o-phenylenediamine: 10 µg/plate for strain: TA 98 (without S9 mix)
9-aminoacridine: 100 µg/plate for strain: TA 1537 (without S9 mix)
4-nitroquinoline-N-oxide: 5 µg/plate for strain: E.coli WP2 uvrA (without S9 mix)
Dissolved in DMSO.

The titer is generally determined only in the experimental parts with S9 mix both for the negative controls and for the two highest doses in all experiments.


Evaluation criteria:
Positive results:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity

Strain

Metabolic activation

Experiment No.

Concentration [µg/plate]

Replicates

Max. factor of revertants

TA 1535

No

No

1

2

20-5000

20-5000

3

3

No relevant increase in the numbers of his+ or trp+ revertans

TA 100

No

No

1

2

20-5000

20-5000

3

3

4.2

4.3

TA 1537

No

No

1

2

20-5000

20-5000

3

3

2.4

2.3

TA 98

No

No

1

2

20-5000

20-5000

3

3

3.0

3.5

E.coli WP2 uvrA

No

No

1

2

20-5000

20-5000

3

3

8.7

9.6

TA 1535

Yes

Yes

1

2

20-5000

20-5000

3

3

No relevant increase in the numbers of his+ or trp+ revertans

TA 100

Yes

Yes

1

2

20-5000

20-5000

3

3

2.8

4.0

TA 1537

Yes

Yes

1

2

20-5000

20-5000

3

3

1.6

2.5

TA 98

Yes

Yes

1

2

20-5000

20-5000

3

3

2.7

3.4

E.coli WP2 uvrA

Yes

Yes

1

2

20-5000

20-5000

3

3

4.0

5.7

Applicant's summary and conclusion

Conclusions:
Thus, under the experimental conditions chosen here, it is concluded that Diethylen-triamine R is a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.